Bifunctional chelating agents

ABSTRACT

Novel homocysteine thiolactone bifunctional chelating agents useful for chelating radionuclides to produce a radiodiagnostic agent for use in in vivo imaging and a method for producing this chelating agent.

This is a division of application Ser. No. 439,960 filed Nov. 8, 1982,now U.S. Pat. No. 4,434,151, issued Feb. 28, 1984.

BACKGROUND OF INVENTION

Scintigraphy and similar radiographic techniques are finding increasingapplication in biological and medical research and diagnosticprocedures. Scintigraphy involves the use of radiopharmaceuticals havinga radioactive material which upon introduction into a biologicalsubject, becomes localized in specific organs, tissue or skeletalmaterial desired to be imaged. When so localized, traces, plates orscintiphotos of the distribution of the radioactive material may be madeby various radiation detectors. The resultant distribution of theradioactive material in the organ or tissue in which it is localized canbe used to detect the presence of abberations, pathological conditionsor the like.

In preparing the radiopharmaceutical, radionuclide chelating agents areutilized which will act as a bridge to connect the radioactive materialsuch as a radioactive metal and the material which will localize in theorgan, or tissue to be imaged. In general, effective chelating agentsare desired which will couple the radionuclides to the material whichwill localize in the organ to be imaged.

SUMMARY OF THE INVENTION

In accordance with this invention, it has been discovered that novelcompounds of the formula: ##STR1## wherein R is hydrogen or lower alkyl,R₁ and R₂ are individually hydrogen or lower alkyl or taken togetherform oxo; R₃ is an amino protecting group where R₁ and R₂ areindividually hydrogen or lower alkyl; R₃ is hydrogen where R₁ and R₂taken together form oxo; R₄ is hydrogen or lower alkyl; R₅ is hydrogenor a thiol protecting group; x and y are integers from O to 2

are bifunctional chelating agents and as such can couple radionuclidesto terminal amino containing compounds capable of localizing in an organor tissue which is desired to be imaged. Hence, the compound of formulaI can be used in preparing radiopharmaceuticals for in vivo diagnosticimaging.

DETAILED DESCRIPTION

The term "lower alkyl" as used throughout this application designatesaliphatic saturated branched or straight chain hydrocarbon monovalentsubstituents containing from 1 to 7 carbon atom such as methyl, ethyl,isopropyl, n-propyl, n-butyl, etc. The term "lower alkoxy" as usedthroughout this specification designates lower alkoxy substituentscontaining from 1 to 7 carbon atoms such as methoxy, ethoxy, isopropoxy,etc. The term "halogen" designates all four halogens such as chlorine,fluorine, iodine, or bromine.

The term "aryl" as utilized herein designates a mononuclear aromatichydrocarbon group which can be unsubstituted or substituted in one ormore positions with a lower alkyl group such as phenyl or tolyl as wellas polynuclear aromatic hydrocarbon groups which can be unsubstituted orsubstituted in one or more positions with a lower alkyl group such asnapthyl, anthryl, phenanthryl, azulyl, etc. The preferred lower arylgroup is phenyl. The term "aryl lower alkyl" designates aryl lower alkylsubstituents where aryl and lower alkyl groups as defined above,particularly benzyl.

The term "lower alkanoyl" as used throughout this specificationdesignates "lower alkanoyl" groups containing from 2 to 7 carbon atomssuch as acetyl, propionyl, etc. The term "arylloweralkanoyl" designatesmonovalent arylloweralkanoyl groups where aryl and lower alkanoyl aredefined as above with phenylacetyl being preferred. The term "aroyl"defines aroyl groups where the aryl group is defined as above withbenzoyl being preferred.

As used herein, the term "thiol protecting group" includes all of theconventional groups which are commonly employed to protect the thiolmoiety. Among these groups are included lower alkylaminocarbonyl such asethylaminocarbonyl, loweralkanoylaminomethyl, aroylaminomethyl, t-butyl,triarylmethyl such as triphenylmethyl, aroyl such as benzoyl,aryloxycarbonyl such as phenoxycarbonyl, arylloweralkoxylcarbonyl,preferably arylmethoxycarbonyl such as benzyloxycarbonyl, loweralkoxycarbonyl such as t-butoxycarbonyl. Among the preferred loweralkanoylaminomethyl groups is aceteamidomethyl and among the preferredaroylaminomethyl is benzoylaminomethyl. The thiol protecting groups areremovable by treatment with heavy metallic ions such as mercuric ions,technetium ions, silver ions, as well as any of the radioactive metalswhich form the complex. Anyof the conventional methods commonly employedin removing these thiol protecting groups can be utilized in accordancewith this invention.

In accordance with this invention, the compound of formula I is preparedfrom a compound of the formula: ##STR2## wherein R, R₁, R₂, R₃, R₄, xand y are as above. When R₁ and R₂ are other than oxo, the nitrogengroup in the compound of formula II is protected with a conventionalamino protecting group to produce a compound of the formula: ##STR3##wherein x, y, R and R₄ are as above; R₆ is an amino protecting group; R₇and R₈ are individually hydrogen or lower alkyl.

Any conventional method of converting a secondary amine to a protectedamine can be utilized in converting the compound of formula II to thecompound of formula II-A. Any of the conventional amino protectinggroups which can be removed by conventional acid hydrolysis or catalytichydrogenation can be utilized in this invention. Among the preferredamino protecting groups are included triarlymethyl such as trityl,arylloweralkoxycarbonyl such as benzyloxycarbonyl, lower alkoxycarbonylsuch as t-butoxycarbonyl, aryl such as benzyl, etc. Any conventionalmethod of preparing these protected amino groups can be utilized inaccordance with this invention. Among these methods are reacting thecompound of formula II where R₁ and R₂ are hydrogen or lower alkyl withthe halide of the protecting group to be introduced into the compound offormula II. Any of the conditions conventional in such reactions can beutilized.

The compounds of formula II or II-A can be either free acids or the acidcan be protected by esterification. The use of an ester increases theyield where either the compound of formulae II or II-A is reacted tointroduce an amino protecting or a thiol protecting group. However, thisester group should be hydrolyzed before the compound of formulae II orII-A is reacted to form the compound of formula I. Any conventionalmethod of ester hydrolysis can be used to form the free acid of formulaI.

A thiol protecting group can be introduced if desired into the compoundof formula II or IIA by conventional means. It has been found that bestresults as far as yields are achieved when the thiol group in thecompound of formula II and formula IIa is protected with any of thegroups hereinbefore mentioned. On the other hand, the reaction andproduction of the bifunctional chelate can be achieved without the useof a thiol protecting group.

The compounds of formulae II or II-A in their free acid form and when R₁and R₂ are hydrogen or a lower alkyl, the amino group is protected withan amino protecting group and which may or may not contain a thiolprotecting group can be converted to the compound of formula I byreaction with a compound of the formula: ##STR4## This reaction iscarried out by conventional amide formation. Any conventional means ofreacting an organic carboxylic acid with an amine to form an amide canbe utilized in this conversion to produce the compound of formula I.Generally, this reaction is carried out in the presence of an amidecondensation agent such as a loweralkylhalo formate, i.e.ethylchloroformate or dicyclohexyl carbodiimide. When utilizing thealkyl chloro formate method, the amide formation occurs by means of amixed anhydride since the alkylchloroformate forms an anhydride with thecompound of formula II-A which then reacts with the amine group on thecompound of formula IV to form the amide of formula I. Any of theconditions conventionally used in reacting an organic acid with an amineto form an amide through the use of a loweralkylhaloformate can beutilized in carrying out this procedure. On the other hand, when acoupling agent such as dicyclohexylcarbodiimide is utilized, any of theconditions conventionally utilized with such coupling agent can be usedto produce the compound of formula I.

The compound of formula I can be converted to a bifunctional anionicchelate of the formula: ##STR5## wherein R₆ is lower alkyl; R₁ and R₂are individually hydrogen or lower alkyl or taken together form oxo; R₄,R, x and y are as above; M is a radioactive metal

via the following intermediates: ##STR6## wherein M, R, R₁, R₂, x, y,R₄, R₄, R₅ and R₆ are as above, with the proviso that R₃ is an aminoprotecting group when R₁ and R₂ are individually hydrogen or loweralkyl; and with the further proviso that R₃ is hydrogen when R₁ and R₂taken together form oxo.

The compound of formula I is converted to the compound of formula VII bytreating the compound of formula I with a base. Any conventional stronginorganic base such as sodium hydroxide, ammonium hydroxide, potassiumhydroxide, etc. can be utilized in carrying out this reaction. Any ofthe conditions conventional in hydrolysis with an alkali metal base canbe used to carry out this reaction.

The compound of formulae I or VII can be converted to the compound offormula VIII by reacting either of these compounds with a compound ofthe formula:

    R.sub.6 --NH.sub.2                                         X

where R₆ is as above.

The compound of formulae VII is reacted with the compound of formula Xutilizing the same conditions described in connection with the reactionof the compound of formula IV with the compound of formulae II or II-Ato produce the compound of formula I. In fact, any conventional methodof condensing a lower alkyl amine with an organic carboxylic acid toproduce an amide can be utilized in carrying out this reaction.

The compound of formula I can also be reacted with the compound offormula X to produce a compound of formula VII. This reaction is carriedout by mixing the compound of formula I and the compound of formula Xtogether in an anhydrous inert organic solvent. Any conventional inertorganic solvent can be utilized in carrying out this reaction with othersolvents such as tetrahydrofuran being preferred. In carrying out thisreaction, room temperature and atmospheric pressure are generallyutilized.

In the next step, the compound of formula VIII where R₃ is an aminoprotecting group is converted to the compound of formula IX by treatingthe compound of formula VIII with an aqueous mineral acid or catalytichydrogenation. Any conventional aqueous mineral acid such as ahydrohalic acid can be utilized in carrying out this reaction to removeprotecting groups which are hydrolyzed by conventional acid hydrolysis.On the other hand when R₃ is an amino protecting group removable byhydrogenation, any conventional method of catalytic hydrogenation can beutilized to remove this protecting group and convert the compound offormula VIII to the compound of formula IX.

The compound of formulae IX where R₁ and R₂ do not form oxo, can be usedto produce the anionic complex of formula VI either as a free base or inthe form of its acid addition salt. Any conventional acid can be used informing these salts. Among these acids are hydrochloric acid, phosphoricacid, acetic acid, propionic acid, citric acid, tartaric acid, etc.

The compound of formula IX is converted to the anionic complex offormula VI by reacting the compound of formula IX with a conventionalsalt of a radioactive metal. In forming the radioactive metal complex offormula X, any conventional radioactive isotope of technetium can beutilized. Among the radioactive isotopes are included technetium-99m.The aforementioned radioactive metals exist with coordination number offive in the complex of formula VI.

The compounds of formula II are known compounds and prepared fromconventional protected amino acids of the formula: ##STR7## wherein Rand y are as above and R₁₀ taken together with its attached oxygen atomform a hydrolyzable ester group such as a lower alkyl ester.

If in the compound of formula II, R₁ and R₂ are oxo, this compound isprepared by reacting the compound of formula XIII with a compound of theformula: ##STR8## where x and R₄ are as above. This reaction is carriedout by amide condensation in the same manner as described in connectionwith the reaction of the compound of formulae II or II-A to produce acompound of formula I.

On the other hand where R₁ and R₂ in the compound of formula I arehydrogen or lower alkyl, the compound of formula II is produced byreacting the compound of formula XV with a compound of the formula##STR9## wherein R₄, R₈ and x are as above; and X is halogen. Thecompound of formula XV is reacted with the compound of formula XIIIutilizing any of the conventional techniques commonly employed inreacting primary amines with halides to produce secondary amines. Inthis manner, the compound of formula II wherein R₁ and R₂ are hydrogenor lower alkyl is produced. If it is desired to produce the compound offormula II in its free acid form, the reaction produced by reacting thecompound of formula XIII with either the compound of formulae XIV or XVis subjected to conventional ester hydrolysis. On the other hand, thethiol group in the compounds of formulae XIV or XV can, if desired, beprotected with a conventional thiol protecting group prior to reactionwith the compound of formula XIII. In this manner, the compound offormula II is produced wherein the thiol group is protected by aconventional thiol protecting group.

In forming the complex of radioactive technetium with the compound offormula IX the technetium complex of formula I, the alkali metal salt oftechnetium-99m pertechnetate is reacted with the compound of formula IXin the presence of a reducing agent such as stannous chloride or sodiumdithionite. The complex of formula VI can be prepared with theconventional salts of radioactive metals of technetium. Among thesesalts are the acetate, citrate and halide salts such as the chloride,bromide, fluoride and iodine salts of these radioactive metals. Amongthe technetium-99m pertechnetate salts are included the alkali metalsalts such as the sodium salts or ammonium salts, or lower alkyl aminesalts. The reaction of the compound of formula IX with the salt of theradioactive metal can be carried out in an aqueous medium at roomtemperature. The anionic complex of formula VI which has a charge of -1can be formed in the aqueous medium in the form of a salt with asuitable cation such as ammonium cation, mono, di or tri-lower alkylamine cation, etc. Any conventional salt of the anionic complex offormula VI with a pharmaceutically acceptable cation can be used inaccordance with this invention. If it is desired to precipitate theanionic complex of formula VI, a salt is formed with a heavy cation suchas tetraphenyl arsinate. Any conventional method of salt formation canbe utilized to produce the chelate of formula VI as a salt. It isthrough the use of a precipitate with a heavy cation that this chelatecan be characterized by structure.

In carrying out the reaction of the compound of formula IX with thesalts of the radioactive metal to form the anionic complex of formulaVI, the thiol protecting group is cleaved. Therefore, this reaction notonly introduces the radioactive metal into the compound of formula VIbut also cleaves the thiol protecting group. All of the aforementionedthiol protecting groups are cleaved by a reaction of salts ofradioactive metals in accordance with this invention.

In forming the complex of formula VI, the radioactive material can haveany suitable amount of radioactivity. In forming the radioactive anioniccomplexes of formula VI, it is generally preferred to form radioactivecomplexes in solutions containing radioactive concentrations of fromabout 0.01 mCi to 100 mCi per ml.

The complex of formula VI can be used for visualizing the organs such asthe kidney for diagnosing disorders in these organs. In accordance withthis invention, the anionic complex of formula I either as a anioniccomplex or as a salt with a pharmaceutically acceptable cation areadministered in a single unit injectable dose. Any of the commoncarriers such as sterile saline solution, plasma, etc., can be utilizedfor preparing the injectable solution to diagnostically image variousorgans in accordance with this invention. Generally, the unit dose to beadministered has a radioactivity of about 0.01 mCi to about 100 mCi,preferably 1 mCi to 20 mCi. The solution to be injected to unit dosageis from about 0.01 ml to about 1 ml. After intravenous administration,imaging of the organ in vivo can take place in a matter of a fewminutes. However, imaging can take place, if desired, in hours or evenlonger, after injecting into patients. In most instances, a sufficientamount of the administered dose will accumulate in the area to be imagedwithin about 0.1 of an hour to permit the taking of scintiphotos. Anyconventional method of imaging for diagnostic purposes can be utilizedin accordance with this invention.

The complexes of formula VI may be administered intravenously in anyconventional medium for intravenous injection such as an aqueous salinemedium, or in blood plasma medium. Such medium may also containconventional pharmaceutical adjunct materials such as, for example,pharmaceutically acceptable salts to adjust the osmotic pressure,buffers, preservatives and the like. Among the preferred mediums arenormal saline and plasma.

The following examples are illustrative but not limitative of theinvention. The percent (%) yields in the following examples are givenbased upon the mols of starting material.

EXAMPLE 1 N-(t-Butyloxycarbonyl)cysteamine-N-acetic acid

Cysteamine-N-acetic acid hydrochloride, 5.16 g (30 mmoles), is dissolvedin 100 mL of deionized water and 10.0 mL (72 mmoles) of freshlydistilled Et₃ N is added.N-t-butyloxycarbonyloxyimino-2-phenylacetonitrile, 7.5 g (30 mmoles), isdissolved in 50 mL of dioxane and added in one portion to the cysteaminesolution. The reaction mixture is stirred at room temperature overnight.The solution is reduced to 50 mL by rotary evaporation and poured into200 mL EtOAc. The EtOAc phase is extracted with 3 portions of 100 mLsaturated NaHCO₃. The NaHCO₃ portions are combined and the pH is loweredto 3.5 by addition of solid citric acid. The yellow product is extractedinto 250 mL of EtOAc. The EtOAc is dried over anhydrous Na₂ SO₄ andremoved by rotary evaporation. After drying under vacuum overnight, 4.25g (60% yield) of the yellow glassy compound,N-(t-butyloxycarbonyl)cysteamine-N-acetic acid, is obtained.

EXAMPLE 2 N-(t-butyloxycarbonyl),N-(2-mercaptoethyl)glycyl homocysteinethiolactone

(a) Mixed Anhydride Method

N-(t-butyloxycarbonyl)cysteamine-N-acetic acid, 1.50 g (6.4 mmoles), isdissolved in 50 mL CH₂ Cl₂ by addition of 1.0 mL (7.2 mmoles) of freshlydistilled Et₃ N. This solution is cooled to 0° C. on an ice bath underan argon atmosphere and 0.85 mL (6.5 mmoles) of isobutylchloroformate isadded dropwise over a period of five minutes. The solution turnsorange-red after 15 minutes of stirring. A solution of 1.16 g (7.5mmoles) of homocysteine thiolactone hydrochloride dissolved in 100 mL ofCH₂ Cl₂ by addition of 2.2 mL (15.8 mmoles) of Et₃ N is added dropwiseover 15 minutes. The solution is stirred at 0° C. for 4 hours and leftto stir at room temperature overnight. The CH₂ Cl₂ solution is extractedwith 2×100 mL of 5% by weight aqueous citric acid, 2×100 mL of saturatedNaHCO₃, and 100 mL saturated NaCl. The CH₂ Cl₂ is dried over anhydrousNa₂ SO₄ and removed by rotary evaporation. After drying under vacuumovernight, 1.5 g (75% yield) of a white glass is obtained. TLC in 5% byvolume methanol-95% by volume chloroform showed the major spot atRf=0.4. The white glass product was purified by silica gelchromatography to yield 0.9 g (45% yield) of N-(t-butyloxycarbonyl),N-(2-mercaptoethyl)glycyl homocysteine thiolactone as a whitecrystalline solid.

(b) Carbodiimide Method

N-(t-butyloxycarbonyl)cysteamine-N-acetic acid, 1.41 g (6 mmoles);1-hydroxybenzotriazole, 1.22 g (9 mmoles); homocysteine thiolactonehydrochloride, 1.0 g (6.5 mmoles); and 2.5 mL (14 mmoles) of freshlydistilled Et₃ N are dissolved in 50 mL of CH₂ Cl₂. The solution iscooled to 0° C. and a solution of 1.24 g (6 mmoles) ofN,N'-dicyclohexylcarbodiimide in 10 mL CH₂ Cl₂ is added in one portion.The solution is stirred on ice for 4 hours and left to stir at roomtemperature overnight. The N,N'-dicyclohexylurea is filtered and the CH₂Cl₂ solution is extracted with 2×100 mL 5% by weight aqueous citricacid, 2×100 mL saturated NaHCO₃, and 100 mL saturated NaCl. The CH₂ Cl₂is dried over anhydrous Na₂ SO₄ and removed by rotary evaporation. Afterdrying under vacuum, 2.1 g of a light pink crystalline product isobtained.

TLC in 5% methanol-95% chloroform showed the major spot at Rf=0.4. Theproduct was purified by silica gel chromatography to yield 0.75 g (37.5%yield) of the white crystalline solid N-(t-butyloxycarbonyl),N-(2-mercaptoethyl)glycyl homocysteine thiolactone.

EXAMPLE 3 N-(t-butyloxycarbonyl), N-(2-mercaptoethyl)glycylN'-methylhomocysteinamide

N-(t-butyloxycarbonyl), N-(2-mercaptoethyl)glycol homocysteinethiolactone, 1.0 g (3 mmoles), is dissolved in 25 mL THF and cooled to0° C. The solution is saturated with methylamine by bubbling methylaminegas through for 10 minutes. The reaction is stirred for 30 minutes andthe solvent removed by rotary evaporation. After drying overnight undervacuum, 1.09 g (100% yield) of N-(t-butyloxycarbonyl),N-(2-mercaptoethyl)glycyl N'-methylhomocysteinamide is obtained as awhite glass.

EXAMPLE 4 N-(2-mercaptoethyl)glycyl N'-methylhomocysteinamidehydrochloride

N-(t-butyloxycarbonyl), N-(2-mercaptoethyl)glycylN'-methylhomocysteinamide, 1.09 g (3 mmoles), is dissolved in 25 mL THFand cooled to 0° C. HCl gas is bubbled through the solution. After oneminute, a white precipitate begins to form. The bubbling is continuedfor 15 minutes and the mixture is stirred for 15 more minutes. The whiteprecipitate is filtered and washed with THF being careful not to dry byair suction. The still wet white product is vacuum dried overnight toyield 550 mg (60% yield) of white, crystalline N-(2-mercaptoethyl)glycylN'-methylhomocysteinamide hydrochloride.

EXAMPLE 5 Tetraphenylarsonium salt of[2-[[1-(2mercaptoethyl)-2-(methylamino)-2-oxoethyl]amino]-N-(2-mercaptoethyl)glycinato-N,N',S,S']oxotechnetate-99

NH₄ ⁹⁹ TcO₄, 50 mg (0.28 mmoles), and N-(2-mercaptoethyl)glycylN'-methylhomocysteinamide hydrochloride, 160 mg (0.53 mmoles), weredissolved in 2 mL of 1:1 parts by volume EtOH and 2N NaOH mixture. Asolution of 50 mg (0.29 mmoles) of Na₂ S₂ O₄ in 1.0 mL 2N NaOH was addeddropwise to the stirred solution turning it from clear to deep orange.Electrophoresis of the orange solution in tris-bartital-sodium barbitalbuffer at pH 8.8 run at 600 V for 45 minutes on paper showed that theorange complex migrated 7.2 cm toward the anode. Electrophoresisindicated a small amount (<1%) of black TcO₂ that stayed at the spottingpoint but showed not TcO₄ ⁻ at its characteristic 12.5 cm migration. Theorange solution was filtered to remove the black, insoluble TcO₂ and wasadded to a solution of 150 mg (0.36 mmoles) of Ph₄ AsCl. H₂ O in 2.0 mLof water. An orange oil separated and was dissolved by adding about 2 mLof methanol. This solution was left to stand in an open beaker forseveral days. After one week orange crystals formed at the bottom of thebeaker and the once dark orange solution was not faintly yellow. Thecrystals were filtered, washed with a small amount of cold waterfollowed by Et₂ O, and dried by air suction to yield 180 mg (86% yield)of tetraphenylarsonium salt of[2-[[1-(2-mercaptoethyl)2-(methylamino)-2-oxoethyl]amino]-N-(2-mercaptoethyl)glycinato-N,N',S,S']oxotechnetate-99as bright orange crystals. Dark orange plate-like crystals suitable forx-ray structure determination were grown by slow evaporation of anethanol-chloroform solution of the product.

EXAMPLE 6[2-[[1-(2mercaptoethyl)-2-(methylamino)-2-oxoethyl]amino]-N-(2-mercaptoethyl)glycinato-N,N',S,S']oxotechnetate-99m

1. Dithionite Reduction at pH 13.3

N-(2-mercaptoethyl)glycyl N'-methylhomocysteinamide hydrochloride, 5 mg(0.17 mmoles), is dissolved in 1.0 mL absolute EtOH and 1.0 mL 1.0NNaOH. A 1.0 mL generator eluant of ^(99m) TcO₄ ⁻ (5 to 50 mCi) in salineis added. Then 0.5 ml of a dithionite solution, prepared by dissolving100 mg Na₂ S₂ O₄ per mL of 1.0N NaOH, is added. After 15-30 minutes, the[2-[[1-(2mercaptoethyl)-2-(methylamino)-2-oxoethyl]amino]-N-(2-mercaptoethyl)glycinato-N,N',S,S']oxotechnetate-99mis prepared [^(99m) Tc-MGHA complex]. This solution of the ^(99m)Tc-MGHA complex is buffered by addition of 1.0 mL of 1N HCl and 4.0 mLof 0.1M NaH₂ PO₄ pH 4.5 buffer. The labeling of this complex isdetermined by electrophoresis in tris-barbital-sodium barbital buffer atpH 8.8 and was run at 600 V for 45 minutes on paper. The ^(99m) Tc-MGHAcomplex migrates 7.2 cm toward the anode under these electrophoreticconditions. The possible impurities, ^(99m) TcO₂ and ^(99m) TcO₄ ⁻, areeasily distinguished from the ^(99m) Tc-MGHA comples by thiselectrophoresis method because the unreduced ^(99m) TcO₄ ⁻ migrates 12.5cm toward the anode while ^(99m) TcO₂ remains at the origin.

2. Stannous Reduction at pH 6.5

N-(2-mercaptoethyl)glycyl N'-methylhomocysteinamide hydrochloride, 5 mg(0.17 mmoles), is dissolved in 1.0 mL EtOH and 1.0 mL 0.1M sodiumacetate at pH 5.5. A 1.0 mL generator eluant of ^(99m) TcO₄ ⁻ (5-50 mCi)in saline is added. Then 0.2 ml of stannous solution, prepared bydissolving 2.0 mg SnCl₂.2H₂ O per mL of ethanol, is added to produce[2-[[1-(2mercaptoethyl)-2-(methylamino)-2-oxoethyl]amino]-N-(2-mercaptoethyl)glycinato-N,N',S,S']oxotechnetate-99m.After 15-30 minutes, the labeling efficiency is determined byelectrophoresis as described under the dithionite reduction method abovewith the same results.

EXAMPLE 7 N-[2-(S-acetamidomethyl)mercaptopropionyl]glycine

2-mercaptopropionylglycine, 20.0 g (122.6 mmoles), andN-hydroxymethyl-acetamide, 12.0 g (134.8 mmoles), are dissolved in 200ml of deionized water. The solution is cooled on an ice bath and 100 mlof conc. HCl is added in one portion. The mixture is stirred on ice forone hour and at room temperature overnight. A white precipitate beginsto form within 1-2 hours.

The reaction mixture is cooled on ice for 4 hours. The white precipitateis filtered, washed with a few mls of ice-cold water, then 2×200 ml Et₂O. The product is dried by air suction for one hour and uncer vacuumovernight to yield 14.4 g (50.2% yield) of the white, crystallineproduct N-[2-(S-acetamidomethyl)mercaptopropionyl]glycine.

EXAMPLE 8 N-[2-(S-benzamidomethyl)mercaptopropionyl]glycine

2-mercaptopropionylglycine, 5.0 g (30.6 mmoles), andN-hydroxymethylbenzamide, 5.0 g (33.1 mmoles), are dissolved in 100 mlof deionized water. The solution is cooled on an ice bath and 50 ml ofconc. aqueous HCl is added in one portion. The mixture is stirred on icefor one hour and at room temperature overnight. A white precipitatebegins to form within thirty minutes. After this period the reactionmixture is cooled on ice for 4 hours. The white precipitate is filtered,washed with ice-cold water, then 2×200 ml Et₂ O. The product is dried byair suction for an hour and under vacuum overnight to yield 8.4 g (94.4%yield) of the white, crystalline productN-[2-(S-benzamidomethyl)mercaptopropionyl]glycine.

EXAMPLE 9 N-[2-(S-acetamidomethyl)mercaptopropionyl]glycyl homocysteinethiolactone

1. Carbodiimide Method

N-[2-(S-acetamidomethyl)mercaptopropionyl]glycine, 2.35 g (10 mmoles),homocysteine thiolactone hydrochloride, 1.55 g (10 mmoles),1-hydroxybenzotriazole, 2.1 g (15 mmoles), and 4.5 ml of Et₃ N aredissolved in 150 ml CH₂ Cl₂. A solution of 2.0 g (9.7 mmoles) ofdicyclohexylcarbodiimide in 50 ml CH₂ Cl₂ is then added in one portionand the mixture is stirred overnight.

The reaction mixture is filtered to remove the white precipitate whichis dicyclohexylurea. The solvent is removed by rotary evaporation. Afterthis, the residue is vacuum dried overnight, and 7.8 g of crude materialremains. The crude product is dissolved in 5 ml of MeOH:CHCl₃ (5/95parts by volume) and loaded on a silica gel column of 8 cm diameter and10 cm height requiring 250 g of silica gel. The column is eluted withMeOH:CHCl₃ (5/95 parts by weight) and 5 ml fractions are collected. Theproduct at R_(f) =0.3 on TLC in MeOH:CHCl₃ (10/90 parts of volume)elutes in fractions 110-190. These fractions are combined and thesolvent is removed by rotary evaporation. After removal of solvent, theresidue is dried under vacuum overnight, 2.0 g (60.6% yield) ofN-[2-S-acetamidomethyl)mercaptopropionyl]glycyl homocysteine thiolactoneas a white glass is obtained.

2. Mixed Anhydride Method

N-[2-(S-acetamidomethyl)mercaptopropionyl]glycine, 7.05 g (30 mmoles),is dissolved in 150 ml CH₂ Cl₂ by addition of 7.5 ml (54 mmoles) of Et₃N. The solution is cooled to -15° C. on a dry ice-acetone bath.Isobutylchloroformate, 3.9 ml (30 mmoles), is added dropwise over a 5minute period. The temperature is maintained at -15° C. throughout theaddition. After the addition of isobutylchloroformate is completed, thereaction is stirred for 3-5 minutes. A solution of 4.65 g (30 mmoles) ofhomocysteine thiolactone hydrochloride and 4.2 ml (30 mmoles) of Et₃ Nin 100 ml of CH₂ Cl₂ is added dropwise over a period of 15-30 minutes ata rate that maintains the temperature at -15° C. The solution is thenstirred at -15° C. for one hour and at room temperature overnight.

The white precipitate that forms is filtered, washed with a small amountof CH₂ Cl₂, dried by air suction for one hour and under vacuumovernight. This yields 3.2 g (32% by weight yield) ofN-[2-(S-acetamidomethyl)mercaptopropionyl]glycyl homocysteinethiolactone as a fine, white powder. TLC on silica gel in MeOH:CHCl₃(10/90 parts by volume) shows a single spot at R_(f) =0.3.

A second crop of N-[2-(S-acetamidomethyl)mercaptopropionyl]glycylhomocysteine thiolactone 700 mg (40% overall yield) of TLC-pure productwas obtained by evaporating the filtrate, dissolving the residue in aminimum of MeOH, and letting the solution stand for two days.

EXAMPLE 10 N-[2-(S-benzamidomethyl)mercaptopropionyl]glycyl homocysteinethiolactone

1. Carbodiimide Method

N-[2-(S-benzamidomethyl)mercaptopropionyl]glycine, 2.96 g (10 mmoles),homocysteine thiolactone hydrochloride, 1.55 g (10 mmoles),1-hydroxybenzotriazole, 2.1 g (15 mmoles), and 4.5 ml Et₃ N is dissolvedin 100 ml CH₂ Cl₂. A solution of 2.0 g (9.7 mmoles) ofdicyclohexylcarbodiimide in 50 ml CH₂ Cl₂ is then added in one portionand the mixture is stirred overnight.

The dicyclohexylurea that precipitates is filtered and the CH₂ Cl₂solution is extracted with 2×200 ml 10% by weight aqueous HCl, 2×200 mlsaturated NaHCO₃, and 200 ml saturated NaCl. The CH₂ Cl₂ layer is driedover anhydrous Na₂ SO₄ and removed by rotary evaporation. The residue isdried under vacuum overnight to yield 2.0 g of a white glass.

The crude product is dissolved in 3.0 ml of MeOH:CHCl₃ (5/95 parts byvolume) and loaded on a silica gel column of 4.5 cm diameter and 12.5 cmheight requiring 100 g of silica gel. The column is eluted withMeOH:CHCl₃ (5/95 parts by volume) and 4 ml fractions are collected. Theproduct at R_(f) =0.4 on TLC in MeOH:CHCl₃ (10/90 parts by volume)elutes in fractions 51-70. The fractions are combined and the solvent isremoved by rotary evaporation. After the residue is dried under vacuumovernight, 1.0 g (26.3% yield) of the white glassN-[2-(S-benzamidomethyl)-mercaptopropionyl]glycyl homocysteinethiolactone is obtained.

2. Mixed Anhydride Method

N-[2-(S-benzamidomethyl)mercaptopropionyl]glycine, 2.96 g (10 mmoles) isdissolved in 100 ml CH₂ Cl₂ by addition of 3.0 ml (22 mmoles) of Et₃ N.The solution is cooled to -15° C. on a dry ice-acetone bath.Isobutylchloroformate, 1.3 ml (10 mmoles), is added dropwise over a 5minute period. The temperature is maintained at -15° C. throughout theaddition. After the addition of isobutylchloroformate is completed, thereaction is stirred for 3-5 minutes. A solution of 1.55 g (10 mmoles) ofhomocysteine thiolactone hydrochloride and 1.4 ml (10 mmoles) of Et₃ Nin 50 ml CH₂ Cl₂ is added dropwise over a period of 15-30 minutes. Thetemperature is maintained at -15° C. throughout the addition. Thesolution is then stirred at -15° C. for one hour and at room temperatureovernight.

The CH₂ Cl₂ solution is washed with 2×200 ml 10% by volume aqueous HCl,2×200 ml saturated NaHCO₃, and 200 ml saturated NaCl. The CH₂ Cl₂ layeris dried over anhydrous Na₂ SO₄ and removed by rotary evaporation. Theresidue is dried under vacuum overnight to yield 2.7 g (71.0% yield) ofN-[2-(S-benzamidomethyl)mercaptopropionyl]glycyl homocysteinethiolactone as a white glass.

EXAMPLE 11 N-[2-(S-acetamidomethyl)mercaptopropionyl]glycylN-methylhomocysteinamide

N-[2-(S-acetamidomethyl)mercaptopropionyl]glycyl homocysteinethiolactone, 1.0 g (3 mmoles), is suspended in 200 ml THF. Methylaminegas is bubbled vigorously through the suspension. Most of the whitestarting material goes into solution in 5-10 minutes. The gas is bubbledfor 15 minutes more at a slow rate. TLC on silica gel in MeOH:CHCl₃(15/85 parts by volume) shows that the reaction is complete. The excessCH₃ NH₂ and also THF is removed by rotary evaporation. After the residueis dried under vacuum overnight, 1.1 g (100% yield) of the white glassN-[2-(S-acetamidomethyl)mercaptopropionyl]glycylN-methylhomocysteinamide is obtained.

EXAMPLE 12

By the procedure of Example 11N-[2-(S-benzamidomethyl)mercaptopropionyl]glycyl homocysteinethiolactone, converted toN-[2-(S-benzamidomethyl)mercaptopropionyl]glycylN-methylhomocysteinamido.

EXAMPLE 13 Tetraphenylarsonium salt of[1-[[1-(2-mercaptoethyl)-2-(methylamino)-2-oxoethyl]amino]-N-(2-mercapto-1-1-oxopropyl)glycinato-N,N',S,S']oxotechnetate-99

NH₄ ⁹⁹ TcO₄, 50.3 mg (0.28 mmoles), and 200.1 mg (0.55 mmoles) ofN-[2-(S-acetamidomethyl)mercaptopropionyl]glycylN-methylhomocysteinamide, were dissolved in 5.0 ml of 1:1 parts ofvolume EtOH:2NNaOH. A solution of 60 mg (0.35 mmoles) Na₂ S₂ O₄ in 1.0ml 2NNaOH was added dropwise. A bright yellow solution resulted and thesolution turned dark orange after sitting for one hour. Electrophoresisof the orange solution was then run in barbital buffer at pH 8.8 at 600V for 45 minutes on paper. The orange complex migrated 6.9 cm toward theanode indicating an anionic complex. No TcO₂ or TcO₄ impurities, whichcome at 0.0 and 12.5 cm, respectively, were observed. A solution of 250mg (0.60 mmoles) of Ph₄ AsCl.H₂ O in 2.0 ml H₂ O was added to the orangesolution and it was left undisturbed overnight. The next day orangeplates had formed at the bottom of the vial and the solution had only alight yellow color. The product was filtered, washed with water, anddried by air suction overnight to yield 158.8 mg (72.2% yield) of thetetraphenylarsonium salt of[1-[[1-(2-mercaptoethyl)-2-(methylamino)-2-oxoethyl]amino]-N-(2-mercapto-1,1-oxopropyl)glycinato-N,N',S,S']oxotechnetate-99as golden-orange plates.

EXAMPLE 14

1. Dithionite Reduction at pH 13

N-[2-(S-acetamidomethyl)mercaptopropionyl]glycylN-methylhomocysteinamide, 20 mg (0.55 mmoles), is dissolved in 2.0 ml1:1 parts by volume of EtOH and 1NNaOH. A 0.5 ml generator eluant of^(99m) TcO₄ ⁻ (5 mCi) is added. Then 0.5 ml of a dithionite solution,prepared by dissolving 100 mg Na₂ S₂ O₄ per ml of 1NNaOH, is added. Thevial is left to stand for 30 minutes. Electrophoresis shows an anioniccomplex which is[1-[[1-(2-mercaptoethyl)-2-(methylamino)-2-oxoethyl]amino]-N-(2-mercapto-1,1-oxopropyl)glycinato-N,N',SS']oxotechnetate-99mand also shows the absence of the possible impurities ^(99m) TcO₂ and^(99m) TcO₄ ⁻.

2. Stannous Reduction at pH 6.5

N-[2-(S-acetamidomethyl)mercaptopropionyl]glycyl homocysteinamide, 20 mg(0.55 mmoles), is dissolved in 1.0 ml 0.1M sodium acetate pH 4.5 bufferand 1.0 ml EtOH. A 0.5 ml generator eluant of ^(99m) TcO₄ -(5 mCi) isadded. Then 0.2 ml of a stannous solution, prepared by dissolving 2.0 mgSnCl₂.2H₂ O per ml EtOH, is added. The vial is left to stand for 30minutes. Electrophoresis shows an anionic complex which is[1-[[1-(2-mercaptoethyl)-2-(methylamino)-2-oxoethyl]amino]-N-(2-mercapto-1-1-oxopropyl)glycinato-N,N',SS']oxotechnetate-99m.

EXAMPLE 15

By both the dithionite reduction at pH 13 procedure in Example 14 andthe stannous reduction at pH 6.5 in Example 14,N-[2-(S-benzamidomethyl)mercaptopropionyl]glycylN-methylhomocysteinamide is converted to[1-[[1-2-mercaptoethyl)-2-(methylamino]-2-oxoethyl]amino]-N-[2-mercapto-1-1-oxopropyl)glycinato-N,N',S,S']oxotechnetate-99m.

EXAMPLE 16

The ^(99m) Tc complex used for the animal studies was prepared inExample 6 following both methods of reduction, stannous chloride ordithionite. The ^(99m) Tc-MGHA complexes labeled by either reductionmethod were tested in animals. The mass concentrations of the ^(99m) Tccomplex administered to test animals were approximately 0.04 mg/kg bodyweight for rabbits and 13.0 mg/kg body weight for mice. Theradioactivity administered to rabbits and mice were about 0.5 mCi and1.5 mCi respectively. The pH of the injectant solution was approximately5 to 6.

Nonanesthetized and nonfasted male rabbits (New Zealand White) weighing3.0-3.5 kg were restrained in dorsal recumbancy approximately 2-3 cmfrom the face of the gamma camera. After intravenous administration of0.1 mL of the ^(99m) Tc-labeled complex solution in the marginal earvein, anterior images were stored by a dedicated computer duringinjection, and 5, 10, 15, 20 and 30 minutes following administration.Relative counts per minute (cpm) versus time curves (uncorrected for^(99m) Tc decay) were determined from quantification of regional areasof interest (RAI) obtained from computer displayed images for ^(99m) Tccomplex.

Nonfasted male mice (1CR strain) weighing 20-24 grams were administeredin the tail vein with 0.2 mL of a ^(99m) Tc-complex solution. At 15, 30,60 minutes post administration groups of four animals were sacrificed bycervical dislocation and selected organs removed for ^(99m) Tc assay ina gross ionization counting chamber. The percentage of the injected dosecorrected for ^(99m) Tc decay was calculated for the various organs ateach sacrifice time.

The RAIs obtained from the gamma camera images for animals administeredwith ⁹⁹ Tc-complex (SnCl₂ reduction method) and ^(99m) Tc-complex(dithionite reduction method) showed significant count densities inregions where the right and left kidneys were visualized. No otherapparent anatomical structures were clearly delineated. The organdistribution of ⁹⁹ m-Tc activity in mice after administration of ^(99m)Tc-complex indicated fast clearance out of the body within the first 15minutes. Subsequent sacrifice times greater than 15 minutes postinjection showed little kidney uptake. Although kidney uptake ofactivity was declining at the various time intervals examined, the totalremaining activity (% injected dose) in each test animal wasapproximately one-half of the injected dose at 30 minutes whichindicated a rapid excretion of activity from the body. This indirectevidence suggests possible kidney clearance of ^(99m) Tc in test animalsadministered ^(99m) Tc-complex.

EXAMPLE 17

The ^(99m) Tc-complex prepared via a dithionite reduction of Example 6was administered to male albino rabbits weighing 3.0-3.5 kg. The drugwas injected into the marginal ear vein. The mass of drug administeredwas equivalent to ˜0.04 mg/kg while the volume injected was 0.1 mL. Theadministered dose was ˜0.5 mCi. Each test animal was restrained indorsal recumbancy approximately 2-3 cm from the face of the gammacamera. Immediately after injection, 100-500 k counts were obtained fromthe gamma camera and stored on floppy disc. The computer controlled themaximum number of counts that could be stored per image. Sequentialimages were obtained and stored on disc at 5, 10, 15, 20 and 30 minutesfollowing administration.

After all images were stored, quantitative image analysis was utilizedto obtain count densities in regional areas of interest (RAI). The RAIswere delineated as distinct anatomical structures. Three distinct areaswere observed. The left and right kidneys and the bladder were clearlydefined.

EXAMPLE 18

A ^(99m) Tc-complex prepared via a stannous chloride reduction ofExample 6 was administtered to male albino rabbits weighing 3.0-3.5 kg.The route of administration and the methods for evaluation of this testdrug are described in Example 17.

EXAMPLE 19

A ^(99m) Tc-MHGA complex prepared via a dithionite reduction of ^(99m)TcO₄ ⁻ of Example 6 was administered to female albino mice weighing20-24 gm. The test drug was administered through the lateral tail veinat a mass dose of approximately 13.0 mg/kg in a volume of 0.2 mL. Thecorresponding radioactive dose administered to each animal was 1.5 mCi.After injection, groups of four animals were sacrificed (cervicaldislocation) at 15, 30 and 60 minutes. The liver, kidney, lung, heart,stomach and intestines (small and large) were removed and assayed for^(99m) Tc activity in a gross ionization counting chamber. Also, thetails and remaining carcasses were individually assayed for ^(99m) Tcactivity.

Knowing the amount of ^(99m) Tc activity administered (assaying for^(99m) Tc in the syringe before and after injection) and the amountassayed in the various organs, the percentage of injected dose (% ID)was calculated as: ##EQU1##

All activities (Ci) were initially corrected for ^(99m) Tc decay back toa fixed starting time using the standard first order differentialequation for decay correction (see below):

    N.sub.1 =N.sub.0 e.sup.-(0.693/T.sbsp.1/2.sup.)t

where N₀ and N₁ are initial and final activities (Ci) respectively. Thehalf-life (T_(1/2)) for ^(99m) Tc decay is 6.02 hr. The value of t isthe elapsed time (hrs.) for the correction.

The results of ^(99m) Tc uptake in various organs and tissues in miceadministered ^(99m) Tc-MGHA labeled via a dithionite reduction is givenin Table 1.

EXAMPLE 20

A ^(99m) Tc-MGHA complex prepared via a stannous chloride reduction of^(99m) TcO₄ ⁻ of Example 6 was administered to female albino miceweighing 20-24 gm. The same methods and procedures given in Example 19were used to calculate the % ID for each organ. The results are given inTable 2.

                  TABLE 1                                                         ______________________________________                                        Organ Uptake of .sup.99m Tc in Mice After Intravenous Administration          of .sup.99m Tc-MGHA (Dithionite Reduction)                                                     (% Injected Dose)                                                             Time After Injection                                         Organ    15 min.      30 min.   60 min.                                       ______________________________________                                        Liver    .sup. 11.3 ± 1.0.sup.1                                                                  7.6 ± .9                                                                             10.2 ± 1.6                                 Spleen   N.D..sup.2   N.D.      N.D.                                          Kidney   3.1 ± .6  3.4 ± .5                                                                             3.9 ± .6                                   Heart    N.D.         N.D.      N.D.                                          Lung      .9 ± .2   .3 ± .1                                                                              .5 ± .3                                   Stomach   .3 ± .01  .1 ± .1                                                                              .8 ± .9                                   Carcass  21.9 ± 2.9                                                                              13.4 ± 5.3                                                                           18.3 ± 2.5                                 Intestines                                                                             16.8 ± 3.0                                                                              20.5 ± 2.0                                                                           16.9 ± 1.4                                 Tail      1.7 ± 1.0                                                                               .3 ± .1                                                                             1.1 ± .4                                   ______________________________________                                         .sup.1 Average of four animals ± standard deviation.                       .sup.2 N.D. = no detectable activity.                                    

                  TABLE 2                                                         ______________________________________                                        Organ Uptake of .sup.99m Tc in Mice After Intravenous Administration          of .sup.99m Tc-MGHA (SnCl.sub.2 Reduction)                                                     (% Injected Dose)                                                             Time After Injection                                         Organ    15 min.      30 min.   60 min.                                       ______________________________________                                        Liver    .sup. 13.3 ± 5.4.sup.1                                                                  6.1 ± .9                                                                             7.7 ± 2.0                                  Spleen   N.D..sup.2   N.D.      N.D.                                          Kidney   3.3 ± .9  1.6 ± .4                                                                             2.3 ± .8                                   Heart    N.D.         N.D.      N.D.                                          Lung      .9 ± .4   .2 ± .2                                                                             .6 ± .3                                    Stomach   .3 ± .4   .6 ± .9                                                                             .1 ± .2                                    Carcass  17.4 ± 2.8                                                                               5.7 ± 2.9                                                                           12.4 ± 5.1                                 Intestines                                                                             28.7 ± 6.1                                                                              27.3 ± 7.0                                                                           29.3 ± 10.4                                Tail     1.4 ± .7   .14 ± .2                                                                            .7 ± .3                                    ______________________________________                                         .sup.1 Average oF four animals ± standard deviation.                       .sup.2 N.D. = No detectable activity.                                    

We claim:
 1. A compound of the formula: ##STR10## wherein R is hydrogenor lower alkyl; R₁ and R₂ are individually hydrogen or lower alkyl ortaken together form oxo; R₃ is an amino protecting group where R₁ and R₂are individually hydrogen or lower alkyl; R₃ is hydrogen when R₁ and R₂taken together form oxo; R₄ is hydrogen or lower alkyl, R₅ is hydrogenor a thiol protecting group; and x and y are integers from 0 to
 2. 2.The compound of claim 1 wherein said compound is N-(t-butyloxycarbonyl),N-(2-mercaptoethyl)glycyl homocysteine thiolactone.
 3. The compound ofclaim 1 wherein said compound isN-[2-(S-acetamidomethyl)mercaptopropionyl]glycyl homocysteinethiolactone.
 4. The compound of claim 1 wherein said compound isN-[2-(S-benzamidomethyl)mercaptopropionyl]glycyl homocysteinethiolactone.